During in vitro cell senescence and many types of tissue differentiation, cells irreversibly withdraw from the cell cycle. Identification of causal or obligatory events in the program leading to irreversible withdrawal has been hampered by the heterogeneous kinetics with which cells in most in vitro systems undergo the transition from the proliferative to post-proliferative phenotype. Myogenic mouse cell lines constitute an excellent model system in which to study this transition because proliferating mouse myoblasts can be induced to rapidly, uniformly and irreversibly enter a post-replicative state by temporary mitogen-depletion. Once basic elements in themechanism of withdrawal are deciphered in the homogeneously behaving mouse myoblast system, they can be tested for their generality in less manageable systems such as in cell senescence. The proosal has 3 specific experimental objectives: 1) Myogenic withdrawal can be triggered by temporary mitogen-depletion. The possibility that senescent withdrawal can be similarly triggered will be investigated by determining whether certain cells in aging human diploid fibroblast-like populations are induced to enter a post-replicative state when temporarily deprived of mitogens. Since, conditions of confluence might mask the existence of a mitogen-depletion effect, human cells will be maintained at clonal densities throughout their in vitro life-span. Determination of life-span at clonal densities has not previously been done. 2) The genetic complexity of the decision to become post-replicative will be investigated by critically evaluating the theory that 5'-bromodeoxy-uridine (BUdR) inhibits myoblast commitment via its effect on a single "dominant acting" gene locus. 3) The dominant or recessive nature of the post-replicative phenotype will be investigated by fusing committed TK- myoblasts and proliferative HPRT- myoblasts and determining whether the committed partner can be restored to a proliferative state. If committed cells can be rescued, an approach using TK- myoblasts and erythrocyte ghost-mediated injections will be developed in an attempt to identify the rescuing factor.